Retinoblastoma, the most common intraocular tumor in children, has been a model system for the study of genetic factors in human oncogenesis. We have cloned, identified, and sequenced the gene conferring susceptibility to human retinoblastoma. Evidence to date indicates a new mode of function of this gene as a suppressor of the cancer phenotype, in that complete inactivation of the gene is required for tumor formation. Based on these important discoveries, we propose four interrelated projects to further explore the role of the retinoblastoma (RB) gene in human oncogenesis. The major aims of these projects are as follows: I. To prepare antibodies recognizing the RB gene product, and to characterize the RB protein biochemically. Antigens have been prepared either from TrypE-RB fusion proteins or from synthesized peptides, and immunized rabbits have yielded antisera to the RB protein. Using one antiserum, a protein of MW 110-114 kD has been identified as the RB gene product. Subcellular localization and DNA binding activity will be assessed to support a possible gene-regulatory activity for the RB protein. II. To elucidate the mechanisms involved in RB gene inactivation. Retinoblastoma tumors may be classified by type of RB gene inactivation, eg. absence of RB expression versus altered RB mRNA size. Studies on one retinoblastoma cell line, Y79, suggest that its two RB alleles have suffered partial DNA deletions and truncation. Natural mutants will be of value in determining specific functional domains of the RB protein. We will attempt to map the most mutable regions of the RB gene; diagnostic probes can be designed according to this map for detection of asymptomatic gene carriers. III. To test tumor-suppressing or promoting activities of normal or mutant RB genes. The normal RB gene will be introduced into retinoblastoma cells by retroviral-mediated gene transfer, and transfected cells tested for loss of tumorigenicity in vivo and reversion of the transformed phenotype in vitro. Complementarily, the RB gene will be specifically inactivated by gene targeting or antisense RNA blocking in cultured cells to test for induction of transformation and/or tumorigenesis. Results to these studies will provide a framework for consideration of gene therapy in cancer. IV. To further characterize the human RB gene locus, and to identify genes related to the RB. The distance between retinoblastoma and esterase D genes will be measured to provide a regional gene map, and flanking genes at the 5' and 3' ends of the RB gene will be isolated. Other putative cancer suppressor genes may be sequence homology to the RB gene and could be isolated by reducing hybridization stringency. Results of these studies will contribute to a basic understanding of retinoblastoma genesis as well as specific clinical applications such as genetic diagnosis. Moreover, this model system holds great promise for further understanding mechanisms of human oncogenesis in general.